Green fluorescent protein as a novel tool to measure apoptosis and necrosis

BACKGROUND: Several apoptosis-detecting methods are currently available. Many of them are work intensive and require the additional use of antibodies, dyes, specific substrates, or enzymatic reactions. A simple, fast, and reliable method was developed to test for apoptosis or necrosis using mouse and human cell lines (e.g., Jurkat, A20.2J, and PB3c cells) stably transfected with a vector coding for green fluorescent protein (GFP) as indicator cells. METHODS: Apoptosis in GFP-transfected cell lines was induced either by soluble Fas-Ligand (sFasL), recombinant human TRAIL (rhTRAIL), or interleukin-3 (IL-3) deprivation. Necrosis was induced by polyclonal anti-A20 and complement treatment of GFP-transfected A20. Cells were analyzed by flow cytometry for GFP fluorescence. Propidium iodide and Annexin V staining were used to confirm the results obtained with the GFP-method. RESULTS: Live GFP-transfected cells show a strong fluorescence intensity, which is significantly diminished upon induction of apoptosis, whereas necrotic GFP-transfected cells almost completely lose their GFP-associated fluorescence. Apoptosis but not necrosis of GFP-transfected cells was blocked by the use of a caspase inhibitor. The results are highly comparable to conventional apoptosis-detecting methods. CONCLUSIONS: The advantage of our GFP-based assay compared with other methods is the analysis of apoptosis or necrosis without the necessity for additional staining or washing steps, making it an ideal tool for screening apoptotic or necrotic stimuli.     

Orthotopic transplantation of rat ovaries as a tool for strain rescue

Spontaneous mutations in inbred strains provide valuable resources for new disease models. Unfortunately, these mutations may affect reproduction, which require considerable efforts in breeding management. We transplanted ovaries of such mutant rat strains orthotopically into ovariectomized premature coisogenic recipients. A reproductive cycle was established in each of the recipients within 5 to 6 weeks after transplantation. Moreover, one-third became pregnant and had litters an average of 3 months after transplantation. These experiments demonstrate that orthotopic transplantation of ovaries can be used in the management of subfertile rat colonies.     

Green fluorescent protein as a secretory reporter and a tool for process optimization in transgenic plant cell cultures

Green fluorescent protein (GFP) is an attractive reporter for bioprocess monitoring. Although expression of GFP in plants has been widely reported, research on the use of GFP in plant cell cultures for bioprocess applications has been limited. In this study, the suitability of GFP as a secretory reporter and a useful tool in plant cell bioprocess optimization was demonstrated. GFP was produced and secreted from suspension cells derived from tobacco that was transformed with a binary vector containing mgfp5-ER cDNA, a modified GFP for efficient sorting to the endoplasmic reticulum, under control of the CaMV 35S promoter. For cell line gfp-13, extracellular and intracellular GFP accumulated to 15.4 and 29.4 mg x 1(-1), respectively. Extracellular GFP accounted for 30.9% of the total extracellular protein. The molecular mass of extracellular GFP was nearly identical to that of a recombinant GFP standard, indicating cleavage of the signal sequence. Neomycin phosphotransferase II, a cytosolic selection marker, was found almost exclusively in cellular extracts with less than 2% in the extracellular medium. These results suggest that extracellular GFP is most likely the result of secretion rather than nonspecific leakage from cells. Furthermore, medium fluorescence intensity correlated nicely with extracellular GFP concentration supporting the use of GFP as a quantitative secretory reporter. During the batch cultivation, culture GFP fluorescence also followed closely with cell growth. A medium feeding strategy was then developed based on culture GFP fluorescence that resulted in improved biomass as well as GFP production in a fed-batch culture.     

Red fluorescent Xenopus laevis: a new tool for grafting analysis

Background  

Fluorescent proteins such as the green fluorescent protein (GFP) have widely been used in transgenic animals as reporter genes. Their use in transgenic Xenopus tadpoles is especially of interest, because large numbers of living animals can easily be screened. To track more than one event in the same animal, fluorescent markers that clearly differ in their emission spectrum are needed.     

Genephony: a knowledge management tool for genome-wide research

Background  

One of the consequences of the rapid and widespread adoption of high-throughput experimental technologies is an exponential increase of the amount of data produced by genome-wide experiments. Researchers increasingly need to handle very large volumes of heterogeneous data, including both the data generated by their own experiments and the data retrieved from publicly available repositories of genomic knowledge. Integration, exploration, manipulation and interpretation of data and information therefore need to become as automated as possible, since their scale and breadth are, in general, beyond the limits of what individual researchers and the basic data management tools in normal use can handle. This paper describes Genephony, a tool we are developing to address these challenges.     

Green fluorescent protein as a marker for monitoring activity of stress-inducible hsp70 rat gene promoter

Murine melanoma cells B16(F10) were stably transfected with a plasmid containing GFP gene linked to rat stress-inducible hsp70.1 gene promoter. Transfected cells show in vitro variable basal levels of fluorescence depending on stress response induced at physiological temperature by growth conditions. Lack of manipulations except medium change resulted in reduction of cellular fluorescence. GFP expression in experimental murine tumors dropped to levels undetectable at physiological temperature. Heat shock induced significant fluorescence of tumor cells both in vitro and in vivo. GFP protein could be a useful marker for studies of mammalian hsp70i gene promoters.     

Green fluorescent protein as a marker for Pseudomonas spp.

The development of sensitive methods for observing individual bacterial cells in a population in experimental models and natural environments, such as in biofilms or on plant roots, is of great importance for studying these systems. We report the construction of plasmids which constitutively express a bright mutant of the green fluorescent protein of the jellyfish Aequorea victoria and are stably maintained in Pseudomonas spp. We demonstrate the utility of these plasmids to detect individual cells in two experimental laboratory systems: (i) the examination of a mixed bacterial population of Pseudomonas aeruginosa and Burkholderia cepacia attached to an abiotic surface and (ii) the association of Pseudomonas fluorescens WCS365 with tomato seedling roots. We also show that two plasmids, pSMC2 and pGB5, are particularly useful, because they are stable in the absence of antibiotic selection, they place an undetectable metabolic burden on cells that carry the plasmids, and cells carrying these constructs continue to fluoresce even after 7 days in culture without the addition of fresh nutrients. The construction of improved Escherichia coli-Pseudomonas shuttle vectors which carry multiple drug resistance markers also is described.     

Green fluorescent protein as a visual marker for wheat transformation

 Wheat (Triticum aestivum L.) transformation via particle bombardment is now established in many laboratories, but transformation efficiencies are still largely low and the highest efficiencies can only be obtained with certain genotypes. For rapid optimization and improvement of wheat transformation protocols, a non-destructive marker which permits early detection of transformed cells is needed. We have assessed the ability of a modified version of the Aequorea victoria green fluorescent protein (GFP) to act as a marker for detecting transformed cells and tissues of wheat. Multicellular clusters emitting green fluorescence were observed 14 days after particle bombardment with a sGFPS65T gene construct, and gfp-expressing shoots (often with expressing roots) could be observed as early as 21 days after bombardment. These shoots can be removed from the callus and grown further until they are ready to transfer to soil. Transgenic wheat plants could be selected on the basis of gfp expression alone although the inclusion of antibiotic resistance as a selectable marker could improve the efficiency. Using sgfpS65T as a marker gene in an experiment comparing bombardment parameters allowed the rapid identification of variables that could be targeted for optimization. Received: 29 March 2000 / Accepted: 29 March 2000     

Non-heart-beating organ donors as a source of kidneys for transplantation: a chart review

BACKGROUND: Organ transplantation is the treatment of choice for patients with end-stage organ failure, but the supply of organs has not increased to meet demand. This study was undertaken to determine the potential for kidney donation from patients with irremediable brain injuries who do not meet the criteria for brain death and who experience cardiopulmonary arrest after withdrawal of ventilatory support (controlled non-heart-beating organ donors). METHODS: The charts of 209 patients who died during 1995 in the Emergency Department and the intensive care unit at the Foothills Hospital in Calgary were reviewed. The records of patients who met the criteria for controlled non-heart-beating organ donation were studied in detail. The main outcome measure was the time from discontinuation of ventilation until cardiopulmonary arrest. RESULTS: Seventeen potential controlled non-heart-beating organ donors were identified. Their mean age was 62 (standard deviation 19) years. Twelve of the patients (71%) had had a cerebrovascular accident, and more than half (10 [59%]) did not meet the criteria for brain death because one or more brain stem reflexes were present. At the time of withdrawal of ventilatory support, the mean serum creatinine level was 71 (29) mumol/L, mean urine output was 214 (178) mL/h, and 9 (53%) patients were receiving inotropic agents. The mean time from withdrawal of ventilatory support to cardiac arrest was 2.3 (5.0) hours; 13 of the 17 patients died within 1 hour, and all but one died within 6 hours. For the year for which charts were reviewed, 33 potential conventional donors (people whose hearts were beating) were identified, of whom 21 (64%) became donors. On the assumption that 40% of the potential controlled non-heart-beating donors would not in fact have been donors (25% because of family refusal and 15% because of nonviability of the organs), there might have been 10 additional donors, which would have increased the supply of cadaveric kidneys for transplantation by 48%. INTERPRETATION: A significant number of viable kidneys could be retrieved and transplanted if eligibility for kidney donation was extended to include controlled non-heart-beating organ donors.     

Green fluorescent protein (GFP) fusion constructs in gene therapy research

The history of green fluorescent protein (GFP) as a marker is less than 10 years old, but it has already made a major impact on many areas of natural sciences, especially on cell biology and histochemistry. GFP can be detected in living cells without selection or staining and it can be fused to other proteins to yield fluorescent chimeras. The potential of GFP has also been recognised by gene therapy researchers and various GFP-tagged therapeutic proteins have been constructed. These chimeric proteins have been used to determine the expression level, site and time course of the therapeutic gene, or the correlation between gene transfer rate and therapeutic outcome. This review summarises the status of the applications of GFP fusions in gene therapy research.     

miRspring: a compact standalone research tool for analyzing miRNA-seq data

High-throughput sequencing for microRNA (miRNA) profiling has revealed a vast complexity of miRNA processing variants, but these are difficult to discern for those without bioinformatics expertise and large computing capability. In this article, we present miRNA Sequence Profiling (miRspring) (http://mirspring.victorchang.edu.au), a software solution that creates a small portable research document that visualizes, calculates and reports on the complexities of miRNA processing. We designed an index-compression algorithm that allows the miRspring document to reproduce a complete miRNA sequence data set while retaining a small file size (typically <3 MB). Through analysis of 73 public data sets, we demonstrate miRspring’s features in assessing quality parameters, miRNA cluster expression levels and miRNA processing. Additionally, we report on a new class of miRNA variants, which we term seed-isomiRs, identified through the novel visualization tools of the miRspring document. Further investigation identified that ∼30% of human miRBase entries are likely to have a seed-isomiR. We believe that miRspring will be a highly useful research tool that will enhance the analysis of miRNA data sets and thus increase our understanding of miRNA biology.     

Green fluorescent protein as a visual selection marker for coffee transformation

The green fluorescent protein (GFP) was used as a visual selectable marker to produce transgenic coffee (Coffea canephora) plants following Agrobacterium-mediated transformation. The binary vector pBECKS 2000.7 containing synthetic gene for GFP (sgfp) S65T and the hygromycin phosphotransferase gene hph both controlled by 35S cauliflower mosaic virus CaMV35S promoters was used for transformation. Embryogenic cultures were initiated from hypocotyls and cotyledon leaves of in vitro grown seedlings and used as target material. Selection of transformed tissue was carried out using GFP visual selection as the sole screen or in combination with a low level of antibiotics (hygromycin 10 mg/L), and the efficiency was compared with antibiotics selection alone (hygromycin 30 mg/L). GFP selection reduced the time for transformed somatic embryos formation from 18 weeks on a hygromycin (30 mg/L) antibiotics containing medium to 8 weeks. Moreover, visual selection of GFP combined with low level of antibiotics selection improved the transformation efficiency and increased the number of transformed coffee plants compared to selection in the presence of antibiotics. Molecular analysis confirmed the presence of the sgfp-S65T coding region in the regenerated plants. Visual screening of transformed cells using GFP by Agrobacterium-mediated transformation techniques was found to be efficient and therefore has the potential for development of selectable marker-free transgenic coffee plants.     

A fluorescent tool set for yeast Atg proteins

Fluorescence microscopy of live cells is instrumental in deciphering the molecular details of autophagy. To facilitate the routine examination of yeast Atg proteins under diverse conditions, here we provide a comprehensive tool set, including (1) plasmids for the expression of GFP chimeras at endogenous levels for most Atg proteins, (2) RFP-Atg8 constructs with improved properties as a PAS marker, and (3) plasmids for the complementation of common yeast auxotrophic markers. We hope that the availability of this tool set will further accelerate yeast autophagy research.     

Green fluorescent protein: A novel viability assay for cryobiological applications

Assessment of tissue viability following the application of a freezing protocol is challenging due to the paucity of viability assays that can be used dynamically, in situ. Cells transfected with a green fluorescent protein (GFP) vector actively produce GFP, which is retained intracellularly. Because of its constitutive and heritable expression, GFP fluorescence of transfected cells may have significant utility as a viability assay for cells within tissues. As a first step toward application to tissues, this work seeks to establish the validity of this GFP-based assay in cell suspensions by comparing the results to other accepted measures of viability. To the authors' knowledge, this is the first use of GFP in cryobiology applications. Intracellular GFP fluorescence was evaluated following slow freezing. Nontransfected and GFP-transfected rat 3230 adenocarcinoma (R3230AC) cells were frozen at 1 degrees C/min to minimum temperatures between -5 and -30 degrees C and then immediately thawed in a 37 degrees C water bath. Samples were assayed using the common viability indicators trypan blue and ethidium bromide (EtBr). A regression analysis of recovery measured with the GFP assay as a function of recovery measured with a trypan blue assay gave a correlation coefficient of 0.97. A similar correlation coefficient, 0.95, was determined for recovery assessed by the GFP assay as a function of recovery measured by an EtBr assay. Nontransfected and GFP-transfected cells responded similarly to slow freezing, indicating that GFP transfection did not significantly alter the response of cells to typical freezing conditions. The excellent correlation of GFP assay results with those of two common viability assays suggests that the GFP-based assay is valid for cells and that further development of a tissue viability assay based on transfection is appropriate.     

Photography as a research tool

This paper attempts to delineate a research design and method for using the photograph as a research tool. An analysis of the photograph's research suitability will consider its ability to structure “articulated visual statements” of the community and to generate “explanatory models” for analysis and interpretation. A significant aspect of the study is the material it offers for a discussion on an important methodological dimension for acquiring knowledge through visual research data. The question is not whether to use photographs as research tools, but how to fit the photograph into the research design and process.     

Green fluorescent protein labeling of primordial germ cells using a nontransgenic method and its application for germ cell transplantation in salmonidae

Transplanting primordial germ cells (PGCs) has a number of potential applications in fish bioengineering. Previously, we established a system to visualize live PGCs in the rainbow trout by introducing the green fluorescent protein (Gfp) gene driven by rainbow trout vasa gene regulatory regions. However, for PGC transplantation to be practically useful in aquaculture, visualization of PGCs using a nontransgenic technique is required. In this study, we demonstrate a method for labeling PGCs from various fish species by introducing chimeric RNAs composed of the Gfp coding region and vasa gene 3'-untranslated regions (UTRs); these sequences play a critical role in stabilizing mRNA in zebrafish PGCs. The GFP chimeric RNAs, including vasa 3'-UTR RNAs from rainbow trout, Nibe croaker, and zebrafish, were microinjected into the cytoplasm of fertilized eggs of several Salmonidae species. All the resulting embryos showed specific labeling in PGCs after the somatogenesis stage, which continued to be visible for at least 50 days. To apply this technique to PGC transplantation, PGCs labeled with chimeric RNA were microinjected into the peritoneal cavity of newly hatched salmonid embryos. The GFP labeling was sufficiently long-lived for the initial stage of donor PGC behavior to be followed in the recipient embryos. Importantly, donor PGCs from brown trout and masu salmon were incorporated into xenogeneic genital ridges in recipient rainbow trout. This nontransgenic method for labeling fish PGCs should be extremely useful for applications of PGC transplantation where the resulting progeny are to be released into the environment, such as PGC cryopreservation for fish stocks and surrogate brood stock technology.     

Green fluorescent antibodies: novel in vitro tools

We produced a fluorescent antibody as a single recombinant protein in Escherichia coli by fusing a red-shifted mutant of green fluorescent protein (EGFP) to a single-chain antibody variable fragment (scFv) specific for hepatitis B surface antigen (HepBsAg). GFP is a cytoplasmic protein and it was not previously known whether it would fold correctly to form a fluorescent protein in the periplasmic space of E.COLI: In this study we showed that EGFP alone or fused to the N'- and C'-termini of the scFv resulted in fusion proteins that were in fact highly fluorescent in the periplasmic space of E.COLI: cells. Further characterization revealed that the periplasmic N'-terminal EGFP-scFv fusion was the most stable form which retained the fluorescent properties of EGFP and the antigen binding properties of the native scFv; thus representing a fully functional chimeric molecule. We also demonstrated the utility of EGFP-scFv in immunofluorescence studies. The results showed positive staining of COS-7 cells transfected with HepBsAg, with comparable sensitivity to a monoclonal antibody or the scFv alone, probed with conventional fluorescein-labelled second antibodies. In this study, we developed a simple technique to produce fluorescent antibodies which can potentially be applied to any scFv. We demonstrated the utility of an EGFP-scFv fusion protein for immunofluorescence studies, but there are many biological systems to which this technology may be applied.